Leucine-rich Repeat Kinase 2 (LRRK2) is a gene belonging to the PARK family of genes. LRRK2 is a complex 285 kD protein containing a RAS of complex GTPase domain (ROC), a C-terminal of ROC domain (COR), and a Ser/Thr kinase domain, in addition to three potential protein interaction domains: an ankyrin domain (ANK), leucine-rich repeats (LRR), and WD40 repeats. Based on the presence of these catalytic domains and scaffold domains, it has been speculated that LRRK2 may be involved in the assembly of multiprotein signaling complexes. (Gardet, A. et al., LRRK2 is involved in the IGN-γ response and host response to pathogens, J Immunol., 2010, 185(9):5577-5585)
The LRRK2 gene is active in the brain and other tissues throughout the body. A number of biological functions have been ascribed to LRRK2, ranging from regulating protein translation through phosphorylating E4-BP, controlling microRNA-mediated translational repression, contributing to neurite extension through interaction with Rac1, regulating autophagy by some unknown mechanism, accelerating neurite degeneration induced by α-synuclein. (Liu, Z. et al., The role of LRRK2 in inflammatory bowel disease, Cell Research, 2012, 22:1092-1094).
The LRRK2 gene provides instructions for making the LRRK2 protein (also called dardarin). One segment of the dardarin protein is called a leucine-rich region because it contains a large amount of a protein building block (amino acid) known as leucine. Proteins with leucine-rich regions appear to play a role in activities that require interactions with other proteins, such as transmitting signals or helping to assemble the cell's structural framework (cytoskeleton). Other parts of the dardarin protein are also thought to be involved in protein-protein interactions.
Additional studies indicate that dardarin has an enzyme function known as kinase activity. Proteins with kinase activity assist in the transfer of a phosphate group (a cluster of oxygen and phosphorus atoms) from the energy molecule ATP to amino acids in certain proteins. This phosphate transfer is called phosphorylation, and it is an essential step in turning on and off many cell activities. Dardarin also has a second enzyme function referred to as a GTPase activity. This activity is associated with a region of the protein called the ROC domain. The ROC domain may help control the overall shape of the dardarin protein.
Parkinson's disease (PD) is the second most common neurodegenerative disease that affects more than 5 million people, accounting to 1-2% of the population worldwide. It is characterized by the loss of dopaminergic neurons in the substantia nigra associated with the formation of fibrillar aggregates that are composed of α-synuclein and other proteins. PD is clinical characterized by four major symptoms; tremor, bradykinesia, rigidity and postural instability. Initially PD was considered sporadic, however genetic studies in patients families revealed mutations that are segregating with PD. In addition to environmental factors, mutations within 6 loci (SNCA, LRRK2, PRKN, DJ1, PINK1 and ATP13A2) have been clearly demonstrated to be causative to familial PD. Among them, SNCA and LRRK2 mutations cause autosomal dominant forms of PD. LRRK2 has been found to be thus far the most frequent cause of late-onset PD. The identification of missense mutations in LRRK2 has redefined the role of genetic variation in PD susceptibility. The mutations are found in 5-6% of patients with familial PD, and also have been implicated with sporadic PD. (F. Y. Ho, K. E. Rosenbusch and A. Kortholt (2014). The Potential of Targeting LRRK2 in Parkinson's Disease, A Synopsis of Parkinson's Disease, Dr. Abdul Qayyum Rana (Ed.), ISBN: 978-953-51-1229-7, InTech, DOI: 10.5772/57362).
Researchers have identified more than 100 LRRK2 gene mutations in families with late-onset Parkinson disease (the most common form of the disorder, which appears after age 50). These mutations replace single amino acids in the dardarin protein, which affects the protein's structure and function. It is unclear how LRRK2 gene mutations lead to the movement and balance problems characteristic of Parkinson disease (PD).
The first two publications of PD associated mutations in LRRK2 described four different pathogenic missense mutations segregating in families of European and North-American origin. Subsequent mutation analyses identified about 80 discrete missense mutations in over a 1,000 families and sporadic patients worldwide. This corresponds to about 50% of all reported unrelated carriers of mutations in the five major genes, making LRRK2 the most frequently mutated PD gene so far. The 80 missense mutations are located over the entire LRRK2 protein and affect all predicted functional domains. Some mutations, though, have much higher frequencies than others, for example, p.Gly2019Ser and mutations altering codon Arg1441. (Nuytemans K., et al., Genetic Etiology of Parkinson Disease Associated with Mutations in SNCA, APRK2, PINK1, PARK7 and LRRK2 Genes: A Mutation Update, Human Mutation, 2010, 31(7): 763-780).
An important observation is that the LRRK2 mutation frequency is seemingly dependent on the ethnicity of the population analyzed. Studies in Caucasians have found that the GLY2019Ser mutation in LRRK2 is a relatively common cause of familial Parkinson's disease and may also be a cause in sporadic Parkinson's disease. For example, the most frequent mutation with a strong founder effect—p.Gly2019Ser—was reported worldwide with an average frequency of 1% in PD patients (Paisan-Ruiz, 2009). But, in Arab Berber and Ashkenazi Jewish populations the p.Gly2019Ser frequency was significantly higher (20 and 40%, respectively) (Lesage et al., 2006; Ozelius et al., 2006), whereas in the first comprehensive screening of a Belgian population, p.Gly2019Ser was apparently absent. (Nuytemans 2010)
A mutation that replaces the amino acid arginine with the amino acid glycine at protein position 1441 (written as Arg1441Gly or R1441G) is a relatively common cause of Parkinson disease in the Basque region between France and Spain. The protein name dardarin comes from the Basque word “dardara,” which means tremor, a characteristic feature of Parkinson disease.
Studies of several different populations from around the world revealed a common LRRK2 gene mutation in 3 to 7 percent of familial Parkinson disease cases. This mutation replaces the amino acid glycine with the amino acid serine at protein position 2019 (written as Gly2019Ser or G2019S). The incidence of the Gly2019Ser mutation in familial cases is highest among Arabs from North Africa and people of Ashkenazi (eastern and central European) Jewish ancestry, and it is lowest in Asian and northern European populations. This particular mutation has also been reported in 1 to 3 percent of sporadic Parkinson disease cases, in which there is no family history of the disease.
Studies in Chinese and Japanese populations have identified an LRRK2 gene mutation that occurs more frequently in people with Parkinson disease than in people without the disease. This mutation replaces the amino acid glycine with the amino acid arginine at protein position 2385 (written as Gly2385Arg or G2385R). This mutation appears to increase the risk of Parkinson disease among people in these populations.
In contrast to other PD genes, LRRK2 mutations have a relatively high frequency of up to 2% in sporadic, late-onset PD patients which makes LRRK2 the most frequently mutated gene of the five major PD genes.
Typically, patients carrying LRRK2 missense mutations present with clinical features similar to those of idiopathic PD, that is, asymmetrical late onset, bradykinesia, rigidity, tremor, and good L-dopa response. The incidence of tremor, however, seems to be elevated in LRRK2 carriers indicating that LRRK2 mutations most likely lead to tremor-dominant disease. (Nuytemans 2010)
It has been suggested that LRRK2 is an IFN-γ target gene that is involved in signaling pathways that are relevant to Crohn's disease (CD) pathogenesis. LRRK2 is highly expressed after IFN-γ stimulation. Crohn's disease is a chronic inflammatory bowel disease (IBD) that is believed to result from dysregulated immune response to commensal intestinal microbiota. The single nucleotide polymorphism (SNP) rs11175593, located in a noncoding region on chromosome 12, is one of the loci identified as a risk factor for CD. LRRK2 expression has been shown to be increased in intestinal tissues upon Crohn's disease inflammation. In inflamed intestinal tissues, LRRK2 is detected in the lamina propria macrophages, B-lymphocytes, and CD103-positive dendritic cells. Furthermore, LRRK2 expression enhances NF-κB-dependent transcription, suggesting its role in immune response signaling. (Gardet 2010)
Recent reports suggest a functional association between LRRK2 and autophagy. The LRRK2 locus has been linked to other diseases such as leprosy and cancer. (Manzoni, C., LRRK2 and autophagy: a common pathway for disease, Biochem Soc Trans, 2012, 40(5): 1147-51)
Leprosy is a chronic infectious and neurological disease that is caused by infection of Mycobacterium leprae (M. leprae). Several LRRK2 variants have been found to be significantly associated with leprosy among the Han Chinese population. (Wang, D. et al., Association of the LRRK2 genetic polymorphisms with leprosy in Han Chinese from Southwest China, Genes Immun., 2015, 16(2):112-9)
An increased incidence of certain non-skin cancers such as renal, breast, lung and prostate cancers, as well as acute myelogenous leukemia (AML), has been reported in Parkinson's disease patients with the LRRK2 G2019S mutation (Saunders-Pullman, R. et al.; Movement Disorders, 2010, 25(15), 2536-2541).
Hexachlorophene (also referred to herein as B10), an organochlorine compound, specifically a chlorinated bisphenol, was widely used as an effective antiseptic for topical applications until the 1970s (Kimbrough, 1971; Pilapil, 1966). It has been found to cause toxicity in animal models (Kimbrough and Gaines, 1971; Thorpe, 1967), and its use was discontinued due to the severity of side effects in human beings. It is generally prepared by the condensation of 2 moles of 2,4,5-trichlorophenol with 1 mole formaldehyde in the presence of concentrated sulfuric acid. B10 can be absorbed into the body through the skin or by ingestion. Oral toxicity studies in rats show an LD50=66 mg/kg.
B10 has been shown to inhibit the Wnt-β-catenin signaling pathway in B lymphoma cells (Min et al., 2009), and recently to inhibit amyloid beta (Aβ) fibril formation and protect primary neuronal cultures from Aβ-induced toxicity (Eleuteri et al., 2014).
In overcoming the toxicity of hexachlorophene (B10) administration alone, another group found that pre-treatment with antioxidants, such as butylated hydroxytoluene (BHT) and ethoxyquin, prior to administration of B10 protects rats from toxicity induced by B10. BHT and ethoxyquin are both free-radical scavengers that prevent lipid peroxidation. In addition, this group also found that agents, such as phenobarbital and SKF-525A (2-diethylaminoethyl-2,2-diphenylvalerate-Hcl), also lessened toxicity when administered prior to B10. The pre-treatment agents were administered daily for at least 3 days prior to administration of B10. It was noteworthy that the protective effect of SKF-525A only occurred upon oral administration of the drug, not ip administration. It was suggested that this effect may occur because a higher SKF-525A is a cytochrome P450 inhibitor which is known to potentiate barbiturate effects and phenobarbital is a barbiturate itself (Hanig, J. P. et al., Protection with butylated hydroxytoluene and other compounds against intoxication and mortality caused by hexachlorophene, Fd Chem. Toxic., 22(3):185-189)
A different group also noted that pre-treatment with the antioxidant L-carnitine also protected rats from the toxicity of B10. (Yapar, K. et al., Protective effects of L-carnitine on the hexachlorophene-induced neurotoxicity and oxidative stress in mice, Revuede Medecine Veterinaire, 2007, 158(12):607-612)
In addition to hexachlorophene, it has been previously found that Heat Shock Protein 90 (Hsp90) inhibitors such as withaferin A (WA) and celestrol also may be used to decrease LRRK2 levels in cells. While both compounds alone reduced LRRK2 levels in cells in a dose and time dependent manner, it was found that treatment with WA in the presence of celastrol enhanced the clearance of LRRK2 in an additive manner. In light of the results, it is suggested that LRRK2 levels can be regulated by targeting the Hsp90-Cdc37 complex. (Narayan, M. et al., Withaferin A regulates LRRK2 levels by interfering with the Hsp90-Cdc37 chaperone complex, Current Aging Science, 2015, 8(1):1-7). Use of Hsp90 inhibitors, such as WA and celastrol, in combination with a drug such as hexachlorophene or its derivatives, could enhance reduction and/or inhibition of LRRK2 protein levels in cells and thus provide a potential treatment for disease associated with LRRK2 mutations.
Given the limited options that exist for treatment of diseases associated with LRRK2 mutations, what is needed is an LRRK2 inhibitor that is capable of reducing LRRK2 expression in patients suffering from diseases associated with LRRK2 mutations or overexpression.